A method for differentiating proteins from nucleic acids in intermediate-resolution density maps: cryo-electron microscopy defines the quaternary structure of the Escherichia coli 70S ribosome

Abstract

Background: This study addresses the general problem of dividing a density map of a nucleic-acid–protein complex obtained by cryo-electron microscopy (cryo-EM) or X-ray crystallography into its two components. When the resolution of the density map approaches ∼3 Å it is generally possible to interpret its shape (i.e., the envelope obtained for a standard choice of threshold) in terms of molecular structure, and assign protein and nucleic acid elements on the basis of their known sequences. The interpretation of low-resolution maps in terms of proteins and nucleic acid elements of known structure is of increasing importance in the study of large macromolecular complexes, but such analyses are difficult. Results: Here we show that it is possible to separate proteins from nucleic acids in a cryo-EM density map, even at 11.5 Å resolution. This is achieved by analysing the (continuous-valued) densities using the difference in scattering density between protein and nucleic acids, the contiguity constraints that the image of any nucleic acid molecule must obey, and the knowledge of the molecular volumes of all proteins. Conclusions: The new method, when applied to an 11.5 Å cryo-EM map of the Escherichia coli 70S ribosome, reproduces boundary assignments between rRNA and proteins made from higher-resolution X-ray maps of the ribosomal subunits with a high degree of accuracy. Plausible predictions for the positions of as yet unassigned proteins and RNA components are also possible. One of the conclusions derived from this separation is that 23S rRNA is solely responsible for the catalysis of peptide bond formation. Application of the separation method to any nucleoprotein complex appears feasible.